Control of human telomerase activity by nucleotide metabolism

Abstract

Telomeres are nucleoprotein structures which flank linear chromosomes and promote genome integrity. Telomere synthesis by the telomerase reverse transcriptase must be tightly controlled to promote cellular function and human health, yet our understanding of the processes which enable telomere length homeostasis is incomplete. Here, I performed the first genome-wide CRISPR-Cas9 screen for telomere length regulating genes in human cells and identified dNTP metabolism as a pathway critical for human telomere length control. Using targeted genetic screening, I found that loss of thymidine nucleotide synthesis or salvage genes led to telomere attrition, whereas loss of the dNTP degrading gene SAMHD1 drove telomere lengthening. Remarkably, I found that supplementation of thymidine nucleoside drove robust telomere lengthening across multiple cell lines, including at doses which had no detectable effects on cell growth, and in a manner which required the presence of telomerase. To further characterize the intersection between dNTP metabolism and telomerase activity control, I developed a method for Telomerase Rapid Assessment in the Cellular Environment, or TRACE. Using TRACE, I identified that guanine nucleotide salvage occupies a critical juncture in telomerase activity control: salvage to form guanosine ribonucleotides drives telomerase inhibition, while salvage to form dGTP stimulates telomerase activation. I propose that dNTP metabolism alters telomere length via the substrate level control of telomerase, adding a new layer to current models of telomerase regulation. Inherited defects in the telomerase components and related genes cause premature telomere shortening and drive a spectrum of fatal disorders called telomere biology disorders (TBDs), manifesting as bone marrow failure, liver and lung fibrosis, and other degenerative diseases throughout the body. There are currently no effective systemic therapies to lengthen telomeres to treat the TBDs, and accordingly the patients diagnosed with these disorders face daunting prognoses. Here, I identify multiple strategies to promote telomere lengthening in TBD patient derived cells by upregulating dNTP metabolism, including supplementation with thymidine, inhibition of the dNTPase SAMHD1, supplementation with deoxyguanosine combined with the PNP inhibitor ulodesine, and expression of an efficient deoxynucleoside kinase from Drosophila melanogaster. Collectively, my work has expanded our understanding of human telomere length control by revealing that telomerase is highly sensitive to manipulations of its dNTP substrates. My findings offer an explanation for emerging human genetic data linking genetic variation in dNTP metabolism genes with telomere length and telomere disease. I propose that the targeted manipulation of dNTP metabolism could provide an avenue to control telomerase activity within a therapeutic window to treat the TBDs. Nucleotide metabolism offers a new lens with which to examine the origins and evolution of telomerase and telomeres.